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  • M E T H O D S I N M O L E C U L A R B I O L O G Y '

    Recombinant Protein Protocols

    Detection and Isolation

    Edited by

    Rocky S. Tuan Thomas Jefferson University, Philadelphia, PA

    Humana Press ^ ^ Totowa, New Jersey

  • © 1997 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

    All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology" is a trademark of The Humana Press Inc.

    All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher.

    This publication is printed on acid-free paper. CgD ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials.

    Cover illustration; Fig. 3B from Chapter 27, "Hyperexpression of a Synthetic Protein-Based Polymer Gene," by Henry Daniell, Chittibabu Guda, David T. McPherson, Xiaorong Zhang, Jie Xu, and Dan W. Urry.

    Cover design by Patricia F. Cleary.

    For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel: 201-256-1699; Fax: 201-256-8341; E-mail:

    Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $8.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-89603-400-3/97 (combbound) 0-89603-48l-X/97 (hardcover) S8.00 -i- $00.25].

    Printed in the United States of America. 1 0 9 8 7 6 5 4 3 2 1

    Library of Congress Cataloging in Publication Data

    Main entry under title:

    Methods in molecular biology™.

    Recombinant protein protocols: detection and isolation/edited by Rocky S. Tuan p. cm.—(Methods in molecular biology'"; vol. 63)

    Includes bibliographical references and index. ISBN 0-89603-400-3 (combbound) (alk. paper); ISBN 0-89603-481-X (hardcover) (alk. Paper) 1. Recombinant proteins—Laboratory manuals. 1. Tuan, Rocky S. II. Series: Methods in molecular

    biology (Totowa, NJ); 63 TP248.65.P76R43 1997 660'.65—dc21 for Library of Congress 97-4023


  • To Cecilia, Chuck, and my parents.

  • Preface

    A major success story of modem molecular biology is the development of technologies to clone and express specific genes. Current applications of recombinant gene products cover a wide spectrum, including gene therapy, production of bioactive pharmaceuticals, synthesis of novel biopolymers, agriculture and animal husbandry, and so on. Inherent in bringing these appli- cations to fruition is the need to design "expression constructs" that will per- mit the ready and specific detection and isolation of the defined recombinant gene products.

    Recombinant Protein Protocols grows out of the need for a laboratory manual on the detection and isolation of recombinantly expressed genes that covers both the background information and the practical laboratory recipes for these analyses. In this book, detailed and contemporary protocols are col- lected to provide the reader with a wide-ranging number of methodologies to enhance the detection and isolation of their gene product(s) of interest. A large number of molecular tags and labels and their usage are described, including enzymes, ligand-binding moieties, immunodetectable molecules, as well as methods to detect interactive proteins, and gene expression-mediated alter- ations in cellular activity. Chapters on in situ detection of gene expression deal with technologies that are currently being applied to the study of gene function and activity. Highlights of applications for recombinant gene expres- sion technologies are provided to give readers exciting perspectives on the future of such technologies.

    Throughout Recombinant Protein Protocols, the authors have consis- tently striven for a balanced presentation of both background information and practical procedures for each of the methodologies treated. The reader is first guided through the necessary supporting background information and then presented with step-by-step specifics for each protocol, including reagents, instrumentation, and other requirements. It is anticipated that this highly practical format, a feature of the Methods in Molecular Biology series, will permit the reader to bring new concepts into personal practice in a most efficient manner.

    The practice of molecular biology as a means to express recombinant genes continues to gain attention in basic biomedical research, as well as the


  • via Preface

    biotechnology and phannaceutical industries. For this reason, it is anticipated that the subjects covered here will continue to be developed, serving as the basis for more sophisticated and efficient methodologies in the future.

    The preparation of Recombinant Protein Protocols would not have been possible without the outstanding work of the contributing authors, all of whom have been most tolerant of my persistent reminders. Dr. John M. Walker, the mastermind of the Methods in Molecular Biology series, was instrumental in initiating and guiding the project. The staff at the Humana Press showed great patience and provided excellent guidance and assistance. My wife, Cecilia, and my newborn son, Chuck, both tolerated my indulgence in the project, and always gave me the necessary emotional support throughout the preparation of the volume. Finally, the excellent secretarial assistance of Margaret Feoli, Susan Lowenstein, and in particular, Lynn Stierle, is gratefully acknowledged.

    Rocky S. Tuan

  • Contents

    Preface vii

    Contents of the Companion Volume xiii

    Contributors xvii


    1 Overview of Experimental Strategies for the Detection and Isolation of Recombinant Proteins and Their Applications Rocky S. Tuan 3


    2 Reporter Systems Debyra J. Groskreutz and Elaine T. Schenborn 11

    3 Detection of Recombinant Protein Based on Reporter Enzyme Activity: ChloramphenicolAcetyltransferase Peiyu Lee and Dennis E. Hruby 31

    4 Human Placental Alkaline Phosphatase as a Marker for Gene Expression Paul Bates and IVIichiael H. Malim 41

    5 Use of Secreted Alkaline Phosphatase as a Reporter of Gene Expression in Mammalian Cells Steven R. Kain 49

    6 Detection of p-Galactosidase and p-Glucuronidase Using Chemiluminescent Reporter Gene Assays Corinne E. M. Olesen, John J. Fortin, John C. Voyta,

    and Irena Bronstein 61 7 Chemiluminescent Immunoassay for the Detection of

    Chloramphenicol Acetyltransferase and Human Growth Hormone Reporter Genes Corinne E. M. Olesen, John C. Voyta, and Irena Bronstein 71

    8 Detection and Selection of Cultured Cells Secreting Recombinant Product by Soft Agar Cloning and Antibody Overlay Marylou G. Gibson, Karmen Hodges, and Lauretta Lowther 77


  • X Contents

    9 Detection and Isolation of Recombinant Protein Based on Binding Affinity Reporter: Maltose Binding Protein Paul Riggs 85

    10 Detection and Isolation of Recombinant Proteins Based on Binding Affinity of Reporter: Protein A Stefan Stahl, Per-Ake Nygren, and Mathias Uhlen 103

    11 Expression and Purification of Recombinant Streptavidin-Containing Chimeric Proteins Takeshi Sano, Cassandra L. Smith, and Charles R. Cantor 119

    12 Bacterial Expression, Purification, and Potential Use of His-Tagged GAL4 Fusion Proteins M. Lienhard Schmitz and Patrick A. Baeuerle 129

    13 Detection of Expressed Recombinant Protein Based on Multidrug Resistance: P-Glycoprotein Ursula A. Germann 139

    14 Detection and Isolation of Recombinant Proteins from Mammalian Cells by Immunoaffinity Chromatography: p53 Jamil Momand and Bahman Sepehrnia 161

    15 Yeast GAL4 Two-Hybrid System: A Genetic System to Identify Proteins Tiiat Interact withi a Target Protein LiZhu 173

    16 Alternative Yeast Two-Hybrid Systems: The Interaction Trap and Interaction l\Aating Erica A. Golemis and Vladimir Khazak 197

    17 Detection of Heterologous G^-Coupled Receptor Activity in LLC-PK^ Cells Based on Expression of Urokinase-Type Plasminogen Activator Luigi Catanzariti and Brian A. Hemmlngs 219


    18 Histochemical and Fluorochrome-Based Detection of p-Galactosidase Ruth Sullivan and Cecilia W. Lo 229

    19 Combined, Sequential In Situ Hybridization and Immunohistochemistry on the Same Tissue Section Kenneth J. Shepley and Rocky S. Tuan 247

    20 Whole Mount In Situ Hybridization to Embryos and Embryonic Tissues Ronald A. Conlon 257

  • Contents xi

    21 Whole-Mount In Situ Hybridization for Developing Chick Embryos Using Digoxygenin-Labeled RNA Probes Christopher W. Hsu and Rocky S. Tuan 263


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